The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2,000 B.C. Until the late 1800s, indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies, and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye's brilliant blue color led to its development as one of the main articles of trade between Europe and the Far East. In 1883, Alfred yon Baeyer identified the chemical structure of indigo: C.sub.16 H.sub.10 N.sub.2 O.sub.2. In 1887, the first commercial chemical manufacturing process for indigo was developed, and is still in use today. This process involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. In the process's final step, indoxyl is then oxidized to indigo by exposure to air.
These commercial chemical processes for manufacturing indigo result not only in production of the dye itself, but also in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic byproducts is desirable. One such environmentally sound method involves indigo biosynthesis by microorganisms.
In a fortuitous discovery, Ensley et al. [(1983) Science, vol. 222, pp: 167-69] found that a DNA fragment from a transmissible plasmid isolated from the soil bacterium Pseudomonas putida enabled Escherichia coli stably transformed with a plasmid harboring the fragment to synthesize indigo in the presence of indole or tryptophan. Ensley et al. postulated that indole, added either as a media supplement or produced as a result enzymatic tryptophan catabolism, was converted to cis-indole-2,3-dihydrodiol and indoxyl by the previously identified multi-subunit enzyme napthalene dioxygenase (NDO) encoded by the P. putida DNA fragment. The indoxyl so produced was then oxidized to indigo upon exposure to air.
NDO had previously been found to catalyze the oxidation of the aromatic hydrocarbon napthalene to (+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronapthalene [Ensley et al., (1982) J. Bact., vol. 149, pp: 948-54]. U.S. Pat. No. 4,520,103, hereby incorporated by reference, describes the microbial production of indigo from indole by an aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprised of multiple subunits: a reductase polypeptide (Rd; molecular weight of approximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxin polypeptide (Fd; molecular weight of approximately 13 kD); and a terminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised of four subunits having an .alpha..sub.2 .beta..sub.2 subunit structure (approximate subunit molecular weights: .alpha., 55 kD; .beta., 21 kD). ISP is known to bind napthalene and in the presence of NADH, Rd, Fd, and oxygen to reduce it to cis-napthalene-dihydrodiol. Fd is the rate-limiting polypeptide in this napthalene oxidation catalysis. See commonly assigned, allowed but not yet issued U.S. patent application Ser. No. 07/389,738, filed Aug. 4, 1989, hereby incorporated by reference, for a thorough discussion of the various NDO subunits and ways to improve them for purposes of indigo biosynthesis.
In addition, aromatic dioxygenases other than NDO may also be useful in the biosynthetic production of indigo. Ensley et al. also observed that a dioxygenase enzyme from another Pseudomonas strain capable of degrading toluene was also able to produce indigo when the culture media was supplemented with indole. For details, see U.S. Pat. No. 4,520,103, supra.
It has also long been known that microorganisms contain biosynthetic pathways for the production of all 20 essential amino acids, including the aromatic amino acid L-tryptophan. The de novo synthesis of aromatic amino acids (phenylalanine, tryptophan, and tyrosine) share a common pathway up through the formation of chorismate. After chorismate synthesis, specific pathways for each of the various aromatic amino acids are employed to complete their synthesis.
Bacterial biosynthesis of tryptophan from chorismate is under the control of the tryptophan (trp) operon. The trp operon, comprised of regulatory regions and five structural genes, has been extensively studied because of its complex and coordinated regulatory systems. The regulatory and structural organization of the trp operon, along with the catalytic activities encoded by the structural genes of the operon, appear in FIG. 1. Of particular relevance to the present invention is the conversion of indole-3'-glycerol-phosphate (InGP), in conjunction with L-serine, to L-tryptophan. The reaction is catalyzed by the multi-subunit enzyme tryptophan synthase (TS). During the reaction, indole is produced as an intermediate. However, the indole is very rapidly combined with L-serine in a stoichiometric fashion to produce L-tryptophan. Thus, no free indole is produced as a result of this InGP plus L-serine conversion to tryptophan.
However, Yanofsky et al. (1959) Proc. Nat'l. Acad. Sci. Vol. 45, pp. 1016-1026, identified a tryptophan synthase mutant which lead to the accumulation of indole. This particular mutant, however, was subject to spontaneous reversion to the wild-type phenotype, as the mutation resulted from a single nucleotide base pair change in a gene coding for one of subunits of tryptophan synthase.
Thus, the goal of the present invention was to create stable tryptophan synthase mutants capable of yielding high levels of intracellular indole. When such indole accumulating routants also express an aromatic dioxygenase enzyme like NDO, this accumulated indole may be converted to indoxyl. Indoxyl so produced may then oxidize to indigo upon exposure to air. Through the commercial application of recombinant DNA technology, a novel and environmentally sound biosynthetic indigo production method has been developed utilizing microorganisms stably transformed with exogenous DNA molecules encoding a modified trp operon and an aromatic dioxygenase enzyme.